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Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus

Co-overexpression of Orc1 and other ORC subunit(s). (A) Estimation of the transfection efficiency. 293T cells were transfected with a plasmid bearing mAG-tagged hRad9 and incubated for 48 h. Microscopic observation was performed, and phase-contrast and fluorescence images are shown. (B) 293T cells were co-transfected with the indicated ORC plasmids and incubated for 48 h. Whole cells were lyzed and analyzed by 9% SDS-PAGE, followed by Western blotting using an anti-Orc1 antibody. Ponceau staining was also performed as a loading control. (C) Isolation of ORC and Orc1–5 by pulldown. Orc1-His, Orc2, Orc3, Orc4, and Orc5 with (+) or without (–) Orc6 were co-overexpressed. Cleared lysates (input; INP) were subjected to a pulldown assay (PD) and analyzed by 9% SDS-PAGE, followed by Coomassie staining. All lanes originate from the same gel.
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Figure 3: Co-overexpression of Orc1 and other ORC subunit(s). (A) Estimation of the transfection efficiency. 293T cells were transfected with a plasmid bearing mAG-tagged hRad9 and incubated for 48 h. Microscopic observation was performed, and phase-contrast and fluorescence images are shown. (B) 293T cells were co-transfected with the indicated ORC plasmids and incubated for 48 h. Whole cells were lyzed and analyzed by 9% SDS-PAGE, followed by Western blotting using an anti-Orc1 antibody. Ponceau staining was also performed as a loading control. (C) Isolation of ORC and Orc1–5 by pulldown. Orc1-His, Orc2, Orc3, Orc4, and Orc5 with (+) or without (–) Orc6 were co-overexpressed. Cleared lysates (input; INP) were subjected to a pulldown assay (PD) and analyzed by 9% SDS-PAGE, followed by Coomassie staining. All lanes originate from the same gel.

Mentions: To test if Orc1 is overexpressed in 293T cells using the constructed plasmid, a series of transient co-expression experiments were performed. A control experiment using a plasmid expressing mAG-tagged hRad9 indicated that most cells were successfully transfected (Figure 3A). Under this condition, expression of His-tagged Orc1 co-transfected with Orc6 was confirmed by Western blotting (Figure 3B). When intact Orc2/3/4/5/6 plasmids were co-transfected with His-tagged Orc1 plasmid, expression of His-tagged Orc1 was also detected at a level similar to that observed when only His-tagged Orc1 plasmid was transfected. Degradation of Orc1 was not observed. Orc1-His and five major proteins corresponding to Orc2/3/4/5/6 were co-pulled down (Figure 3C), suggesting that all of the ORC subunits were co-overexpressed and formed a complex. Although Uno et al. successfully co-transfected up to three plasmids simultaneously (Uno and Masai, 2011; Uno et al., 2012), our data demonstrated that co-transfection of six plasmids was tolerable for co-overexpression using this system. Hereafter, 0.26 μg of each plasmid per 10-cm plate was used during the course of this study.


Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Kawakami H, Ohashi E, Tsurimoto T, Katayama T - Front Microbiol (2016)

Co-overexpression of Orc1 and other ORC subunit(s). (A) Estimation of the transfection efficiency. 293T cells were transfected with a plasmid bearing mAG-tagged hRad9 and incubated for 48 h. Microscopic observation was performed, and phase-contrast and fluorescence images are shown. (B) 293T cells were co-transfected with the indicated ORC plasmids and incubated for 48 h. Whole cells were lyzed and analyzed by 9% SDS-PAGE, followed by Western blotting using an anti-Orc1 antibody. Ponceau staining was also performed as a loading control. (C) Isolation of ORC and Orc1–5 by pulldown. Orc1-His, Orc2, Orc3, Orc4, and Orc5 with (+) or without (–) Orc6 were co-overexpressed. Cleared lysates (input; INP) were subjected to a pulldown assay (PD) and analyzed by 9% SDS-PAGE, followed by Coomassie staining. All lanes originate from the same gel.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834435&req=5

Figure 3: Co-overexpression of Orc1 and other ORC subunit(s). (A) Estimation of the transfection efficiency. 293T cells were transfected with a plasmid bearing mAG-tagged hRad9 and incubated for 48 h. Microscopic observation was performed, and phase-contrast and fluorescence images are shown. (B) 293T cells were co-transfected with the indicated ORC plasmids and incubated for 48 h. Whole cells were lyzed and analyzed by 9% SDS-PAGE, followed by Western blotting using an anti-Orc1 antibody. Ponceau staining was also performed as a loading control. (C) Isolation of ORC and Orc1–5 by pulldown. Orc1-His, Orc2, Orc3, Orc4, and Orc5 with (+) or without (–) Orc6 were co-overexpressed. Cleared lysates (input; INP) were subjected to a pulldown assay (PD) and analyzed by 9% SDS-PAGE, followed by Coomassie staining. All lanes originate from the same gel.
Mentions: To test if Orc1 is overexpressed in 293T cells using the constructed plasmid, a series of transient co-expression experiments were performed. A control experiment using a plasmid expressing mAG-tagged hRad9 indicated that most cells were successfully transfected (Figure 3A). Under this condition, expression of His-tagged Orc1 co-transfected with Orc6 was confirmed by Western blotting (Figure 3B). When intact Orc2/3/4/5/6 plasmids were co-transfected with His-tagged Orc1 plasmid, expression of His-tagged Orc1 was also detected at a level similar to that observed when only His-tagged Orc1 plasmid was transfected. Degradation of Orc1 was not observed. Orc1-His and five major proteins corresponding to Orc2/3/4/5/6 were co-pulled down (Figure 3C), suggesting that all of the ORC subunits were co-overexpressed and formed a complex. Although Uno et al. successfully co-transfected up to three plasmids simultaneously (Uno and Masai, 2011; Uno et al., 2012), our data demonstrated that co-transfection of six plasmids was tolerable for co-overexpression using this system. Hereafter, 0.26 μg of each plasmid per 10-cm plate was used during the course of this study.

Bottom Line: All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex.Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells.The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan.

ABSTRACT
Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

No MeSH data available.


Related in: MedlinePlus